Review Reports - Salivary NETosis-Related and Oxidative Stress Biomarkers Define a Conventional Cigarette Smoking-Associated Inflammatory Phenotype in Periodontitis: A Cross-Sectional Observational Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses a clinically relevant topic and has several strengths, including the inclusion of former smokers and biochemical verification of smoking status by salivary cotinine. The study is generally well organized, and the findings are of potential interest. However, several issues would benefit from clarification before publication.

Major comments

Although the authors adjusted for stage and grade, current smokers appear to have substantially more severe periodontitis than never-smokers, including higher PPD, CAL, PISA, and a greater prevalence of severe disease categories. Therefore, some of the biomarker differences may still reflect differences in inflammatory burden and tissue destruction rather than smoking exposure alone. The Stage III sensitivity analysis is helpful, but the interpretation would be stronger with a more cautious discussion of possible residual confounding.

The selected biomarker panel is interesting, but some markers are indirect and not fully specific for NET formation. In particular, cfDNA is nonspecific, neutrophil elastase may reflect processes other than NETosis, and salivary measurements may be influenced by multiple oral sources. The manuscript would benefit from a clearer discussion of these limitations and from slightly more cautious wording, such as “NETosis-related biomarkers” rather than direct statements implying specific measurement of NETosis activity.

The manuscript acknowledges the limitations of its cross-sectional design, which is appropriate. However, some statements could still be interpreted as mechanistic or near-causal. I would encourage the authors to ensure that the abstract, discussion, and conclusion consistently frame the findings as associations.

The composite NETosis and oxidative-inflammatory scores are potentially useful, but their construction would benefit from additional explanation. It would be helpful to clarify the rationale for the averaging approach and to provide more detail on whether internal consistency, dimensional structure, or collinearity among component biomarkers was examined.

Minor comments

The discussion could further expand on the limitations of salivary biomarkers, including the influence of whole-mouth inflammation and other oral factors.

Please clarify whether clinical periodontal examiners were blinded to smoking status, or whether this was not feasible.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is generally well written and methodologically detailed; however, the novelty should be more clearly defined because several recent studies have already described smoking-associated NETosis and oxidative-inflammatory alterations in periodontitis.

Since the study is cross-sectional, the manuscript should refrain from making causal or mechanistic claims that smoking directly causes the observed biomarker changes or that quitting smoking results in biological recovery.
Current smokers demonstrated substantially more severe periodontal disease than the other groups, including higher PPD, CAL, PISA, and a greater prevalence of Stage III and Grade C periodontitis, which raises concern that disease severity itself may partly explain the biomarker differences.
Although the authors adjusted for stage and grade and performed sensitivity analyses, residual confounding by disease severity likely remains and should be acknowledged more explicitly as a study limitation.
The manuscript appears to overstate the specificity of MPO-DNA complexes and related biomarkers as indicators of NETosis, as these markers may also reflect broader inflammatory or cellular damage processes in saliva.
The limitations of saliva as a biological matrix should be discussed more critically because salivary biomarkers may originate from multiple oral and systemic sources rather than specifically from periodontal NET formation.
The methodology used to construct the composite NETosis and oxidative-inflammatory scores requires further justification, particularly given the extremely large reported effect sizes.
The authors should clarify whether internal consistency testing, multicollinearity assessment, or dimensionality reduction analyses were performed before constructing the composite scores.
The translational claims regarding the clinical applicability of these salivary biomarkers should be moderated because no diagnostic performance analyses, predictive validation, or longitudinal assessments were performed.
The absence of microbiome analysis is a significant limitation, as smoking-associated alterations in the subgingival microbiota could substantially contribute to the observed inflammatory phenotype.
The manuscript would benefit from a clearer description of participant recruitment procedures, including whether consecutive or convenience sampling was used.
The authors should justify whether a 2-hour abstinence period from smoking before saliva collection is sufficient to minimise acute smoking-related effects on inflammatory and oxidative biomarkers.
The reported salivary cotinine concentrations in current smokers exceeded the stated assay working range, and therefore, additional clarification regarding sample dilution and assay linearity is required.
The discussion should be revised to consistently frame the findings as associations rather than evidence of smoking-driven biological modulation.